Success in identifying a homologue of a prototype gene rests on several factors. Foremost is the rate of sequence divergence, which will determine whether sufficient remnants of a primordial sequence are retained over time to enable cloning and identification of homologous genes over great evolutionary distances.
For example, if sequence similarity between the prototype gene in one organism and its homologue in another organism (which is to be cloned) is relatively high, extending across much of the molecule, then one expects any part of the prototype sequence to provide an adequate probe to detect the homologue.
Further, even if sequence similarity is not high, one may still be able to clone the homologue by subdividing the prototype gene into smaller fragments and test each for its binding to DNA of the target organism. However, only fragments that harbor conserved DNA sequence elements are expected to detect the homologue. An alternative approach to the development of probes depends on the design of degenerate oligonucleotides. This approach requires a consensus protein sequence derived from analysis of multiple homologues of the desired gene. Such analysis appears essential for successful long distance homology cloning.
Long distance homology cloning pertains to periods of time in excess of 600-700 million years. Though the method described in this invention illustrates how to clone across phyla of metazoan organisms, it can be applied to longer time frames or to crossing phyla within other kingdoms, such as plants or fungi. A useful point of reference pertinent to metazoans is when the coelomic lineage, which led to both protostomes and deuterostomes, separated from pseudocoelomates and acoelomic metazoans.
The present invention addresses the central question in long distance homology cloning; namely, what steps are necessary in order to develop probes capable of discriminating homologues from non-homologous DNA?